In-house efforts have led to development and implementation of a small molecule microarray onto which a library of 20,000 small molecules has been printed. Fluorescently-labeled RNAs ( 60 nucleotides in length) are flowed over the array, and binding is recorded as a fluorescent signal. Importantly, the entire array can be probed within one week, and is sparing on RNA amounts. Using this system, we have successfully identified novel chemotypes that recognize the HIV-1 trans-activation response (TAR) element in biochemical assays, and inhibit virus replication in culture. Medicinal chemistry was successful in deriving novel chemotypes, whose binding site was identified by NMR spectroscopy. This pilot project has been the basis for expanded efforts to target regulatory RNAS with small molecules. A second HIV cis-acting RNA under investigation is the Rev response element (RRE), which is important for nucleocytoplasmic transport of the viral genome. Stem-loop IIB (SLIIB) of the RRE provides the primary binding site for the HIV Rev protein, and recent analysis of post-transcriptional modification of the HIV-1 genome has revealed two potential sites of modification in RRE SLIIB. We extended such observations to ask whether m6A-modified RNA provides a unique signal for small molecule recognition. Indeed, using our small molecule microarray, chemotypes that recognized non-methylated or m6A-methylated RRE SLIIB were identified, in addition to a third class that recognized both RNAs. Understanding the nature of conformational changes induced by m6A modification with respect to selective small molecule recognition is underway. Applying the small molecule microarray strategy to the ENE element of KSHV PAN lncRNA has identified chemotypes that recognize the triple helix. Biological testing indicated that a subset of these compounds was not cytotoxic, which has allowed us to investigate their effect on KSHV replication. Unexpectedly, one compound was capable of reactivating KSHV from latency, and the molecular mechanism is presently under investigation. More importantly, our discovery of a novel chemotype that activates KSV from latency opens the possibility of developing a kick-and-kill strategy whereby activated virus can be targeted by a second set of small molecules targeted to specific viral enzymes. As will be outlined later, a parallel project in the laboratory has indeed identified small molecules that target one or more HSHV nucleotidyltransferases. We have also considered the option that antagonizing triple helix formation might be considered a therapeutic strategy. A duplex version of the ENE was therefore investigated, which has revealed additional, unique chemotypes. Biological testing of these compounds is planned for the near future. This translational project is combined with biochemical (SHAPE-MaP) and biophysical approaches (NMR, SAXS) to provide high resolution information on ENE/small molecule complexes that can be used for structure-based drug design. Since the screening strategy we have designed requires covalent linkage of small molecule to the microscope slide, this has the potential to mask the pharmacophore. In order to address this shortcoming, we have recently developed a solution-based screening strategy that is more amenable to the extensive collection of small molecules curated by the NCI Molecular Targets Laboratory (MTL), and in particular its vast collection of natural product extracts. Using stem-loop A (SLA) of the Dengue virus RNA genome as a model system, we have developed a low-cost, robust and reproducible high throughput thermal denaturation assay (HTS-Thermofluor), with which novel chemotypes were identified from the MTL collection of pure natural products. We have also shown that the same strategy can be applied to the MTL collection of natural product extracts, which represent a unique collection of novel chemical entities. Subsequent identification of SLA-binding chemotypes will be accomplished by LC/MS. A full screen of 250,000 molecules requires 20 mg of purified RNA, and, through its commitment to NMR analysis of regulatory RNAs, the laboratory has sufficient capacity for high level RNA production and purification. A unique feature of KSHV PAN is that it is present in the nucleus, cytoplasm and purified virions. In collaboration with researchers of the NCI ACVP, we have completed a structural analysis of the 1200-nt PAN in each of these cellular compartment, a goal of which was to reveal alterations on PAN occupancy by viral and/or cellular proteins. This study, recently published in Nucleic Acids Research, was extended to map the sites of recombinant KSHV proteins on in vitro transcribed PAN RNA.